中国2个地方品系猪SLA-3原核表达载体构建及表达

doi:10.16431/j.cnki.1671-7236.2015.02.009

›› 2015, Vol. 42 ›› Issue (2): 298-302.doi: 10.16431/j.cnki.1671-7236.2015.02.009

中国2个地方品系猪SLA-3原核表达载体构建及表达

苟东洲, 戴琳, 杨杰, 高凤山

大连大学生命科学与技术学院, 大连 116622

收稿日期:2014-09-04 出版日期:2015-02-20 发布日期:2015-02-13
通讯作者: 高凤山 E-mail:gfsh0626@126.com
作者简介:苟东洲(1993-),男,甘肃定西人,学士,研究方向:生物工程,E-mail:2363079807@qq.com
基金资助:
国家自然科学基金项目(31172304);2014年辽宁省大学生创新创业训练计划项目(201411258068);2013年大连大学大学生创新创业训练计划项目(2013071)

Construction and Expression of the Prokaryotic Expressing Vector of Pigs SLA-3 Derived from Two Chinese Local Strains

GOU Dong-zhou, DAI Lin, YANG Jie, GAO Feng-shan

College of Life Science and Technology, Dalian University, Dalian 116622, China

Received:2014-09-04 Online:2015-02-20 Published:2015-02-13

摘要/Abstract

摘要： 为构建中国地方品系荷包猪及莱芜黑猪SLA-3(命名为SLA-3-HB和SLA-3-LW)原核表达载体及表达目的蛋白,试验通过PCR扩增获得SLA-3胞外区基因,并克隆至pMD19-T Simple载体,转化大肠杆菌Top10感受态细胞,酶切及测序筛选阳性重组质粒;重组质粒经酶切回收,目的片段进一步连接pET-21a(+)表达载体,并转化大肠杆菌BL21感受态细胞,IPTG诱导目的基因的表达;SDS-PAGE检测目的蛋白。结果显示,PCR成功扩增SLA-3-HB及SLA-3-LW胞外区,大小约850 bp,目的基因成功克隆至pMD19-T Simple载体,并筛选序列正确的阳性重组质粒。进一步研究证实,SLA-3-HB及SLA-3-LW成功连接到表达载体pET-21a(+),插入片段长度均为831 bp。经诱导后,SLA-3-HB及SLA-3-LW均成功表达,表达蛋白大小为31 ku,相对表达含量达到40%。本研究成功构建了中国地方品系荷包猪及莱芜黑猪SLA-3原核表达载体,为进一步研究其结构和功能奠定基础。

关键词: 荷包猪; 莱芜黑猪; SLA-3; 表达

Abstract: To construct the prokaryotic expressing vector of SLA-3 derived from Hebao and Laiwu pigs (named as SLA-3-HB and SLA-3-LW) and to express the interest of protein,the extracellular domain of SLA-3 were amplified by PCR,and then they were cloned into the pMD19-T Simple vector followed by transforming into Escherichia coli Top10.By cleavage and sequencing,the positive clones were selected.After cleavage and purification of the recombinant plasmids,the interest of genes were further ligated to pET-21a(+) and transformed into the Escherichia coli BL21.The interest of genes were induced to express by IPTG and the interest of proteins were detected by SDS-PAGE.The results showed that the extracellular domain of SLA-3-HB and SLA-3-LW were successfully amplified with the molecular weight of 850 bp.It was also shown that the SLA-3-HB and SLA-3-LW were successfully cloned into pMD19-T Simple vector and the positive recombinant plasmids with correct sequences were selected.Further study proved that SLA-3-HB and SLA-3-LW were successfully ligated into pET-21a(+) with the 831 bp inserted fragments.After induction,SLA-3-HB and SLA-3-LW were all expressed successfully and the molecular weight of the interest of proteins were about 31 ku,and the contents of the expression of the proteins reached 40 percent.In this study,the prokaryotic expression vector of SLA-3 gene derived from Hebao and Laiwu pigs,which would lay a base for furter study the structure and function of the interest of proteins.

Key words: Hebao pig; Laiwu pig; SLA-3; expression

中图分类号:

苟东洲, 戴琳, 杨杰, 高凤山. 中国2个地方品系猪SLA-3原核表达载体构建及表达[J]. , 2015, 42(2): 298-302.

GOU Dong-zhou, DAI Lin, YANG Jie, GAO Feng-shan. Construction and Expression of the Prokaryotic Expressing Vector of Pigs SLA-3 Derived from Two Chinese Local Strains[J]. , 2015, 42(2): 298-302.

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中国2个地方品系猪SLA-3原核表达载体构建及表达

Construction and Expression of the Prokaryotic Expressing Vector of Pigs SLA-3 Derived from Two Chinese Local Strains

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